The aim of this proposal is to examine the production of the cholera toxin (CT)-like enterotoxin synthesized by some strains of non-01 V. cholerae and to purify it and compare its structure and function with that of cholera toxin. Strains of non-01 V. cholerae isolated from human diarrheas which produce CT-like toxin in amounts similar to freshly isolated V. cholerae E1 Tor will be used. Optimal culture conditions for production of toxin will be determined by the method of response surfaces and the distribution of CT-like toxin in relation to the cell will be compared to that in classical and E1 Tor V. cholerae. An attempt will be made to increase toxin yield by mutagenizing cells with nitrosoguanidine or by exposing cultures to lincomycin. Purification schemes will be based initially on those used successfully in purifying CT (e.g., phosphocellulose chromatography) and toxin will be monitored using assays developed for CT, which have already been shown to measure the non-01 toxin as well. The subunit structure of purified CT-like toxin will be examined using SDS-PAGE. If similar to those of CT, subunits will be isolated by gel chromatography under dissociating conditions or by phosphocellulose chromatography. Purified materials will be compared to CT holotoxin and its A and b subunits using electrofocusing, PAGE and SDS-PAGE, amino acid analysis and immunodiffusion. The techniques and reagents developed will then be used to examine the distribution of CT-like toxin and subunits among other biovars of non-01 V. cholerae.